Edwardsiella piscicida is found to be an important facultative intracellular pathogen with a broad host range. These organisms can replicate and survive within host macrophages to escape from the subversion of the immune defense. E. piscicida-macrophage interaction is very important in determining the outcome of edwardsiellasis. As an effector protein of E. piscicida T6SS, EvpP has been determined to be a very important virulence factor for E. piscicida, although its precise role in E. piscicida-macrophage interactions is not yet clear. In this study, the roles of EvpP in E. piscicida-macrophage interactions were characterized. Here, we constructed the deletion mutants of evpP (ΔevpP) and complementation (ΔevpP-C) by the allelic exchange method. Compared to wild type strain (WT), ΔevpP was found to be attenuated for growth within macrophages. In line with this observation, we found its survival capacity was lower than WT under oxidative and acid stress in vitro, which simulate conditions encountered in host macrophages. Attenuation of ΔevpP also correlated with enhanced activation of macrophages, as reflected by augmented NO production in ΔevpP-treated macrophages. Moreover, compared to WT, ΔevpP induced markedly increased apoptosis of macrophages, characterized by increased Annexin V binding and the activation of cleaved caspase-3. These findings provided strong evidence that EvpP is involved in the process of E. piscicida-macrophage interactions and is required for its survival and replication in macrophages. Thus, we propose that EvpP might be an important factor that controlling the fate of E. piscicida inside macrophages. To further exploring the underlying mechanism of EvpP action, the cDNA library was constructed from E. piscicida-infected macrophages and a yeast two-hybrid screen was performed to search for cellular proteins interacting with EvpP. Ribosomal protein S5 (RPS5) was identified as a target of EvpP. Furthermore, the interaction was validated with co-immunoprecipitation assay. This result implies that the observed effect of EvpP on macrophages might be related to RPS5-mediated regulation, contributing to a better understanding of the mechanisms of EvpP involved in E. piscicida-macrophage interactions. Edwardsiella piscicida is pathogen bacterium that could infect a wide range of hosts from fish to amphibians, bird, reptiles, and mammals including humans  was cellular proteins which could interact with EvpP. This work could increase our understanding of the diversity of EvpP functions involved in E. piscicida-macrophage interactions.Although EvpP is involved in pathogenesis of E. piscicida strain QL-S (GenBank no. DQ233654) were stored at −70°C with 15% glycerol. Bacterial cultures were grown in TSA or TSB . RAW264.7 cells were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences and cultured in Dulbecco's modified Eagle's medium (DMEM) . The cells were passaged twice a week to keep an exponential growth.E. piscicida with primer MF1/MR1 and MF2/MR2, respectively . To assay the effects of acidification, H2O2, high-osmolarity stress and heat treatment, the cells were incubated at pH 3.0 for 15 min, in the presence of 440 mM H2O2 for 40 min, in the presence of 1.5 M NaCl for 20 min, and at 50°C for 60 min, respectively. The bacteria were serially diluted and spread on TSA for CFU determination. Each experiment was performed in triplicate.All the tested strains were cultured to the stationary growth phase in TSB  of 50. At 12 and 24 h post-infection, the level of NO was determined by measuring the amount of nitrite in the culture medium using a NO detection Kit . The optical density of each well was determined using a microplate reader with an emission wave length at 550 nm.6 cells/mL. The cell suspension was stained with 5 μL Annexin V-FITC and 10 μL propidium iodide  for 15 min at room temperatures in the dark. Quantification of apoptotic cells was analysis by flow cytometry . The percentage of apoptotic cells was defined as the sum of early and late apoptotic cells.Infections were conducted as described above for the macrophage survival assay. At the end the 24 and 48 h post-incubation, cells were collected and resuspended in binding buffer at density of 1 × 10Infection experiment were conducted as described above for the apoptosis assay. Following infection, cells were lysed for western blot analysis. Briefly, equal amounts of protein from samples were separated on a 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane. GAPDH antibody  was used as the loading control. After being blocked with 5% BSA, the membrane was incubated overnight with caspase-3 antibody . Following extensive washing, the membrane was then incubated with appropriate secondary antibodies for 2 h at room temperature. The proteins were visualized with Western Lightning™ Chemiluminescence Reagent Plus kit . The relative protein expression intensities were quantified by densitometry using Labworks™ Analysis Software . Each reaction was performed in triplicate.The RAW264.7 cells were treated as described above for the intracellular replication experiment. At the end of 5 h post-incubation, total RNA was isolated for cDNA library construction. Make Your Own “Mate & Plate™” Library system  was used to establish cDNA library as described by Cai et al. .evpP was synthesized and cloned into pGBKT7 to generate pGBKT7-evpP as a bait plasmid. In addition, evpP was cloned into pcDNA3.1 (+) with a Myc-tag to generate Myc-evpP. The RPS5 gene was amplified and inserted into pcDNA3.1 (+) with a HA-tag to create HA-RPS5. The validity of all vectors was verified by enzymatic digestion and DNA sequencing.The full-length sequence of evpP plasmid and empty pGBKT7 vector were transformed into the yeast strain Y2HGold using theYeast maker™ Yeast Transformation System 2 kit according to the manufacturer's manual , respectively. Total proteins were detected using c-myc tag mouse McAb  to detect evpP expression as described previously  plates. The clones were then transferred to SD/-Leu-Trp-His-Ade/X/A (QDO/X/A) plates three times. The blue colonies on QDO/X/A agar plates were confirmed as positive hits and the corresponding prey plasmids were sequenced and BLAST-aligned using NCBI. To eliminate false positive hits, prey plasmids was co-transformed with pGBKT7-evpP into Y2HGold cells and the co-transformants were grown on QDO/X/A plates. Co-transformation with BD-p53/AD-T and BD-Lam/AD-T served as positive and negative controls, respectively.Following elimination of autoactivation and toxicity of EvpP, Y2HGold transformed with pGBKT7- evpP and 5 μg HA- Rps5 (Co-transfection with Myc and HA-Rps5 or Myc- evpP and HA as a control). At 48 h post-incubation, the cells were lysed and the 80% of supernatant was subjected to co-IP experiments. The lysate was immunoprecipitated with anti-HA antibody  followed by western blot analysis. The inverse coimmunoprecipitation experiment was also performed with anti-Myc antibody . Briefly, mouse anti-HA , mouse anti-myc  or normal mouse IgG control antibody  were separately added into the protein lysate, and incubated overnight at 4°C, followed by adding 30 μL Protein A/G magnetic beads  and incubating overnight at 4°C. Subsequently, the beads were gently washed by centrifugation 3 times and subjected to western blot analysis with rabbit anti-Myc  and rabbit anti-HA antibody , respectively. Input assays were performed using the remaining 20% of the supernatant for analysis of HA-Rps5 and Myc- evpP protein expression. All experiments were repeated at least three times independently.To validate the EvpP-Rps5 interaction discovered using the two-hybrid system in yeast, we performed coimmunoprecipitation (Co-IP) experiments in 293T cell line. Cells were grown until 80% confluence and co-transfected with 5 μg Myc- P < 0.05 was considered statistically significant throughout the study.All data are expressed as the mean ± standard deviation (SD) and statistically analyzed using one-way ANOVA followed by Tukey's Multiple Comparison test. E. piscicida, evpP deletion mutant (ΔevpP) and complementation ΔevpP (ΔevpP-C) were constructed. We compared the growth of these three strains under cultured conditions to exam whether the deletion of the evpP affected the growth of E. piscicida in vitro. The growth curves demonstrated that there was no notable difference in the growth rate of ΔevpP and WT  (Accession no.: NM_009095.2). Other 4 genes were ruled out for its very low sequence homology. To confirm the specificity of the interaction, prey plasmids were co-transformed with pGBKT7-evpP into Y2HGold cells, resulting in blue colonies growth on QDO/X/A plates. Co-transformations with BD-p53/AD-T and BD-Lam/AD-T as positive, and negative controls, respectively, indicated that the experiments were successful  is a target of EvpP. Furthermore, the interaction was validated with co-immunoprecipitation assay, indicating the reliability of the Y2H approach. These results imply that EvpP might exert an influence on host macrophages by means of interaction with RPS5. RPS5, as an important component of ribosomes, forms part of the exit site on the small ribosomal subunit and cross-links to the exit-site tRNA (Matragkou et al., To further exploring the underlying mechanism of EvpP exerting effect on macrophages, the cDNA library was constructed from E. piscicida-induced apoptosis warrant further investigation. In addition to its role involved in apoptosis, RPS5 has been implicated in performing function involved in signal transduction. RPS5 could participate in Akt signaling pathway and were found to reduces Akt Phosphorylation (Xu et al., E. piscicida-infected macrophages. It is well-known that increasing production of NO is closely related to activation of NF-κB signaling pathway (Akira and Takeda, It is worthwhile to note that RPS5 has been demonstrated to possess important other extra-ribosomal functions including apoptosis and signal transduction (Vizirianakis et al., 2+-dependent MAPK-Jnk pathway in E. piscicida-infected macrophages (Chen et al., 2+ signaling is still unclear. Perhaps, the screen of target proteins interacting with EvpP in our study will contribute to explaining how EvpP regulates intracellular Ca2+ signaling. Given the prominent role of RPS5 in cell signaling transduction, our findings imply that effects of EvpP on intracellular Ca2+ signaling in macrophages might be achieved by PRS5-mediated signal pathway, although the precise mechanism of its linkage to RPS5 is remains unclear. Combined with our research results, the finding of pyroptosis in E. piscicida-infected macrophages revealed that EvpP might offer a double protective function for E. piscicida survival in macrophages via its anti-pyroptosis and anti-apoptosis properties. Nevertheless, in present study, it is noteworthy that more early apoptosis could be observed than late apoptosis in E. piscicida-infected macrophages, implying that cell death by apoptosis might play a significant role in E. piscicida-macrophage interactions. Although EvpP have been demonstrated to exert inhibitory effect on apoptosis and pyroptosis in E. piscicida-infected macrophages, for E. ictalurid, EvpP were shown to increase apoptosis and necrosis in anterior kidney macrophages (Kalindamar et al., E. piscicida, deletion of evpP were not found to affect the virulence of E. ictalurid significantly in that study. These contrasting observations remain to be explained, implicating that EvpP might play diverse roles in different pathogens confronting different cellular hosts.Interestingly, EvpP was reported to prevent the NLPR3 inflammasome activation-mediated pyroptosis by inhibiting intracellular CaE. piscicida-macrophages interaction. Our results indicate that EvpP of E. piscicida is required for its survival and replication in macrophages. Furthermore, we revealed that EvpP interacts with RPS5 in infected macrophages providing insight into the function of EvpP. However, further investigation relating to the EvpP-RPS5 interaction and the function of RPS5 in E. piscicida infection will be performed in a future study.Taken together, our study uncovered a significant role of EvpP associated with The datasets generated for this study are available on request to the corresponding author.LQ conceived the planning of the study, design of experiments, mentored the researchers, wrote the paper, and contributed to some of the experiments. XW, YG, and WW conducted the experiments. KB contributed the technical assistance.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In the partnership between the medical departments of Würzburg University, Germany, and Nagasaki University, Japan, palliative care is a relevant topic. The aim of the study was to perform a comparative analysis of the hospital-based palliative care teams in Würzburg (PCT-W) and Nagasaki (PCT-N). Survey of staff composition and retrospective analysis of PCT patient charts in both PCTs were conducted. Patients self-assessed their symptoms in PCT-W and in Radiation Oncology Würzburg (RO-W). The (negative) quality indicator ‘percentage of deceased hospitalised patients with PCT contact for less than 3 days before death’ :505–509, 2005) was analysed. Both PCTs follow a multidisciplinary team approach. PCT-N saw 410 cancer patients versus 853 patients for PCT-W (22.8% non-cancer patients). The Eastern Cooperative Oncology Group Performance Status at first contact with PCT-N was 3 or 4 in 39.3% of patients versus 79.0% for PCT-W. PCT-N was engaged in co-management longer than PCT-W . The most frequent patient-reported psychological symptom was anxiety , followed by depression (98.2% PCT-W and 80.3% RO-W). In 14 of the 148 deceased patients, PCT-N contact was initiated less than 3 days before death (9.4%) versus 121 of the 729 deceased PCT-W patients (16.6%). Psychological needs are highly relevant in both Germany and Japan, with more than 85% anxiety and depression in patients in the Japanese IPOS validation study (Sakurai in Jpn J Clin Oncol 49(3):257–262, 2019). This should be taken into account when implementing PCTs. As part of joint research activities between the medical departments of Nagasaki University and Würzburg University that started in 2011, our focus was set on palliative care. In the spirit and tradition of the famous Würzburg-born physician and Japanologist Philipp Franz von Siebold (1796–1866) who used his stay in Nagasaki between 1823 and 1829 to teach Japanese colleagues about Western medicine and to learn about Japanese science and culture, a fruitful exchange of practical and personal experience was made possible by visiting the palliative care units in both university hospitals and sharing opinions as well as knowledge gained from our respective experiences.A closer look at the facts and figures revealed similarities as well as differences, and this comparison might be a useful new starting point for reflecting upon ‘traditional’, possibly entrenched, convictions and for discussing practical consequences of or within the respective existing systems, especially as those ‘grown structures’ are the result of both different historical developments and cultural backgrounds that can be evaluated better by considering the outcome of different solutions to similar problems in different countries.Textbook of pastoral medicine (1878), should be mentioned.It is widely accepted that palliative care was not ‘invented’ by the hospice movement in the 1960s Stolberg /2017 andCancer has been the leading cause of death in Japan since 1981. About 370,000 people died in Japan from cancer in 2017. After heart failure, cancer is the second most frequent cause of death in Germany. In Germany, about 235,700 people died of cancer in 2017. Regarding the history of modern palliative care, it was especially initiated in the case of cancer patients and their families. Multidisciplinary non-profit societies for palliative medicine were funded in Germany in 1994 and in Japan in 1996. In the last few decades, great efforts have been made in both countries to improve palliative care starting with initiatives to improve palliative care especially for cancer patients nearing end of life. Several laws and programmes supported the implementation of palliative care  are well established; in University Hospital Würzburg, these are supplemented by a palliative care unit with ten beds. In 2019, an exchange about the work of the two hospital-based palliative care teams began. Hospital-based palliative care teams have a tradition going back to the 1980s, starting in the UK, the USA, Canada and Australia  is recommended for the demand-oriented integration of multidisciplinary specialised palliative care into cancer patients’ medical care, based on the complexity of care regarding patient-reported needs, problems with care, patients’ general conditions and the Australian casemix classification for palliative care  and PCT Nagasaki (PCT-N) with a special focus on team composition and working mode, the clinical characteristics of patients, symptom burden and contents of care, and on the integration of the PCTs into end-of-life care in the two hospitals. Because only PCT-W assessed patient-reported concern, in the discussion the Würzburg data are compared to the data from the Japanese validation study .The IPOS integrates the most important patient-reported symptoms and concerns in a 17-item multidimensional tool examining physical and psychiatric symptoms as well as communication, spiritual and practical issues  ‘percentage of deceased hospitalised patients with PCT contact’ and (2) ‘percentage of patients with PCT contact for less than 3 days before death’ were analysed. These quality indicators were first presented by Earle et al. for benchmarking. They can be assessed very easily using the charts of the deceased hospitalised patients. The proposed expert-approved quality goals are 55% for PCT contact before death and less than 8% for PCT contact for less than 3 days before death for all patients with PCT contact , while PCT-W saw 853 patients (22.8% of them were non-cancer patients). The mean age of the Nagasaki patients was 67 years (median 64.7) versus 69 years (median 68.4 years) for the Würzburg patients. 59.0% of Nagasaki patients and 52.8% of Würzburg patients were male. The ECOG Performance Status of the Nagasaki patients at first contact with the PCCS was 0–1 in 34.1%, 2 in 26.6%, 3 in 21.5% and 4 in 17.8% versus 0–1 in 6.1%, 2 in 14.9%, 3 in 40.8% and 4 in 38.2% for the Würzberg patients. Seventy-seven patients who died in hospital were co-managed by PCT-N (18.8%) versus 106 patients who were co-managed by PCT-W (12.4%). See Table Between January and September 2019, 498 patients in PCT-W filled in the IPOS in its entirety. Between May to December 2018, 549 cancer patients filled in the IPOS in the University Hospital Würzburg’s Department of Radiation Oncology during the waiting time before first contact with the radiation oncologist (RO-W). 174 of them were treated with palliative intent and 375 with curative/adjuvant intent . Anxiety was the most cited symptom , followed by depression (98.2% PCT-W and 75.7% RO-W), not feeling at peace (97.0% PCT-W and 63.0% RO-W), poor mobility (95.1% PCT-W and 77.1% RO-W), poor appetite (90.4% PCT-W and 33.3% RO-W), practical problems (85.2% PCT-W and 91.4% RO-W), not sharing feelings (84.1% PCT-W and 66.8% RO-W), dry mouth (78.0% PCT-W and 40.0% RO-W), pain (72.1% PCT-W and 62.2% RO-W), information needs (69.4% PCT-W and 75.0% RO-W), shortness of breath (57.0% PCT-W and 43.2% RO-W), constipation (56.3% PCT-W and 28.2% RO-W), nausea (26.3% PCT-W and 24.7% RO-W) and vomiting (25.6% PCT-W and 8.3% RO-W); see also Fig. PCT-N was engaged in palliative care co-management for longer than PCT-W  versus 121 of the 729 deceased PCT patients in Würzburg (16.6%). If only the cancer patients are considered, there were notably more deceased patients co-managed by SPC  in the USA as well as in the German Hospice and Palliative Registry , different core sets of data for the benchmarking of PCTs were defined  in radiation oncology, 72% of curatively and 86% of palliatively treated patients reported at least one clinically relevant physical or psychological symptom, mostly decreased general well-being , followed by tiredness (35% of curative patients) and pain , followed by insomnia/disturbed sleep (48%), pain (48%), dry mouth (47%) and anorexia/appetite changes (45%). They propose the use of a core set of symptoms that also include ‘depression’ and ‘difficulty concentrating/remembering’ for assessment across clinical trials and in the routine of cancer care, particularly among patients with advanced diseases (Reilly et al. Over 90% of the PCT-W patients reported one or more psychiatric symptoms or psychosocial needs and more than 50% reported one or more physical symptoms. This is higher than that reported in the Japanese validation study of the IPOS (142 adult patients in palliative care units in six hospitals, Sakurai et al. Unfortunately, further comparison is not possible, because in the international literature the threshold values for symptom prevalence are defined as any IPOS symptoms/problems specified as ‘moderate’, ‘severe’ or ‘overwhelming’ and not the ‘slight’ or higher threshold that was used by Sakurai et al. and in the Würzburg analysis. In the IPOS validation study, patients in German and British palliative care units or under PCT co-management were interviewed (392 participants, 77.7% cancer patients, Murtagh and Ramsenthaler Regarding psychological symptoms and needs, 86–97% of patients in our samples reported anxiety or depression. This is considerably more than in the analysis of Reilly and colleagues (34% depression/sadness, no information given regarding anxiety, Reilly et al. The S3-LL Guidelines recommend the use of validated screening tools for the detection of depression and anxiety such as the ultra-brief Patient Health Questionnaire for Depression and Anxiety (PHQ4, Löwe et al. In view of the frequency of the two symptoms, it seems important to integrate psychiatric and/or psycho-oncologic expertise into PCTs so that these issues can be addressed professionally. The Japanese team composition has a clear advantage here compared to the German situation where psychiatrists or psycho-oncologists are not recommended in a PCT. 82–98% of patients reported family anxiety due to the patient’s situation. This underlines the importance of holistic palliative care for relatives as well as for patients.Pain is a relevant problem, but not one of the most commonly reported symptoms. In our study, 62–72% of patients reported pain versus 66% of Japanese palliative cancer patients (Sakurai et al. On the other hand, good pain management is in most cases easier than interventions and helps patients to cope with fatigue, weakness or poor mobility. A contemporary review shows that fatigue can be managed with different types of exercise in combination with psycho-education and other non-pharmacological interventions (Hilfiker et al. We used the adapted quality indicator ‘percentage of hospitalised cancer patients with PCT contact 3 days or more before death’ to evaluate the integration of SPC into inpatient end-of-life care. Both PCTs did not achieve the quality goals defined by Earle et al.  of 55% oThe thresholds for the quality indicators were expert approved more than 15 years ago. Until now, there has been intensive debate about the right cutoff for quality indicators evaluating SPC integration in hospital and in general. The analysis of Dasch et al.  shows thThere is an international consensus that inpatient end-of-life care has to be performed by the primary teams together with the palliative care specialists. A structured needs-based approach is recommended because it can provide quality of life for patients more successfully than a consultation-based approach (van Mechelen et al. Our study has several limitations. It is retrospective and cultural aspects as well as aspects of the different health-care systems are neglected. The database is small and a predefined consensus about the documented items is missing. Therefore, only descriptive analysis was performed. Neither PCT-W nor PCT-N document their treatment strategies or patient-reported outcome parameters. Therefore, we used a quality indicator based on administrative data to describe the integration of the two PCTs into inpatient end-of-life care. For a planned prospective joint project, the documentation will first be harmonised, based on national and international standards (Guo et al. This exploratory analysis showed that the organisation and work processes of the two PCTs as well as the problems of the patients are similar. In contrast to the public image of palliative care, it is psychosocial needs, not pain, that are mostly cited by patients. In further studies, patient- or relative-reported outcomes should be assessed in addition to administrative data and treatment strategies. An in-depth analysis of cultural aspects and of the different health-care systems should also be part of further comparative analyses.


Cellular therapy based on mesenchymal stem cells (MSCs) is a promising novel therapeutic strategy for the osteonecrosis of the femoral head (ONFH), which is gradually becoming popular, particularly for early-stage ONFH. Nonetheless, the MSC-based therapy is challenging due to certain limitations, such as limited self-renewal capability of cells, availability of donor MSCs, and the costs involved in donor screening. As an alternative approach, MSCs derived from induced pluripotent stem cells (iPSCs), which may lead to further standardized-cell preparations.n = 16) and patients with the fracture of the femoral neck (n = 12) were obtained during operation. The bone marrow-derived MSCs (BMSCs) were isolated by density gradient centrifugation. BMSCs of ONFH patients (ONFH-BMSCs) were reprogrammed to iPSCs, following which the iPSCs were differentiated into MSCs (iPSC-MSCs). Forty adult male rats were randomly divided into following groups (n = 10 per group): (a) normal control group, (b) methylprednisolone (MPS) group, (c) MPS + BMSCs treated group, and (d) MPS + iPSC-MSC-treated group. Eight weeks after the establishment of the ONFH model, rats in BMSC-treated group and iPSC-MSC-treated group were implanted with BMSCs and iPSC-MSCs through intrabone marrow injection. Bone repair of the femoral head necrosis area was analyzed after MSC transplantation.In the present study, the bone marrow samples of patients with ONFH (The morphology, immunophenotype, in vitro differentiation potential, and DNA methylation patterns of iPSC-MSCs were similar to those of normal BMSCs, while the proliferation of iPSC-MSCs was higher and no tumorigenic ability was exhibited. Furthermore, comparing the effectiveness of iPSC-MSCs and the normal BMSCs in an ONFH rat model revealed that the iPSC-MSCs was equivalent to normal BMSCs in preventing bone loss and promoting bone repair in the necrosis region of the femoral head.Reprogramming can reverse the abnormal proliferation, differentiation, and DNA methylation patterns of ONFH-BMSCs. Transplantation of iPSC-MSCs could effectively promote bone repair and angiogenesis in the necrosis area of the femoral head.The online version contains supplementary material available at 10.1186/s13287-021-02249-1. Osteonecrosis of the femoral head (ONFH) is a progressive and refractory disease that is common in orthopedics. Although ONFH is not fatal, it is persistently painful, seriously affecting the quality of life of the patients. The usual cause of ONFH is the inadequate blood supply to the trabecular bone in the femoral head, ultimately leading to bone cell death due to various factors, such as excessive alcohol consumption, hormone application, trauma, and diseases of connective tissue, which is followed by the collapse of the articular cartilage and subsequent osteoarthritis –6. AccorBMSCs are a kind of adult stem cells, which exhibit the ability of self-renewal and multilineage differentiation under specific conditions , 17. BMSStem cell-derived MSCs have emerged as an important alternative source for BMSCs, providing a feasible solution to the above-stated problems. Previously, human embryonic stem cells (ESCs) were commonly used as a source of MSCs. However, the extensive application of ESC-MSCs in the clinical intervention was limited greatly due to concerns related to ethical issues and immunogenicity. A potentially inexhaustible cell resource with a pluripotency potential similar to the ESCs is represented by the induced pluripotent stem cells (iPSCs) generated from autologous somatic cells. The iPSCs present the potential for patient-specific autologous therapies, thereby eliminating most of the problems associated with immune incompatibility and ethical issues, and introducing a novel direction for regenerative medicine based on cell transplantation . In addiWith the approval of the ethics committee, as stated previously, a total of 28 bone marrow donors were recruited in the present study. The recruited participants were the patients who underwent total hip replacement between May 2016 and November 2017, at the Wuhan Union Hospital, Huazhong University of Science and Technology. Among these 28 patients, 16 were diagnosed with ONFH based on the medical imaging and histopathology results. The bone marrow samples (10 mL each) for the experiments were obtained during surgery. The bone marrow samples from the 12 patients with fractured femoral neck served as the source of normal control cells. The clinical characteristics of the participating patients are summarized in Supporting Information Table S1.5 cells/cm2 and incubated at 37 °C with 5% CO2. The non-adherent cells were removed after 3 days of culture, and the medium was changed every 2 days. When 80–90% confluence was reached, the cells were harvested using 0.25% trypsin containing 0.02% EDTA , and were reseeded at the ratio of 1:3 for initial subculture. After three passages, the BMSCs were ready for the subsequent experiments.The BMSCs derived from the ONFH patients (ONFH-BMSCs) and the control BMSCs were isolated and cultured, as reported previously . Briefly6 cells/mL. The following antibodies were used in the analysis: mouse anti-human CD44-PE, CD13-PE, CD73-APC, CD34-PE, CD105-FITC, and HLA-DR-FITC . The cells were analyzed under the Flow Cytometry Caliber instrument . BD Diva software version 6.1.3 was used for data collection and analysis . Briefly, the cells were suspended in phosphate-buffered saline (PBS) at a concentration of 104 cells/well in a 6-well plate and cultured for 24 h. After starved with DMEM medium for 2 h, cells were transduced with equal amounts of four concentrated viruses supplemented with 8 μg/ml polybrene . The day was considered as day 0. Transduction was repeated after 2 days. On day 6, cells were harvested with 0.25% trypsin-EDTA, and 5 × 104 cells were seeded on mouse embryonic fibroblasts (MEFs) feeder in a gelatin-coated 100 mm culture dish cultured with mTeSR-1 medium . The medium was changed every other day. ES-like colonies usually appeared from day 13. About on day 20–22, the ES-like colonies were mechanically isolated. Every single picked colony was cultured and expanded in a Matrigel-coated 24-well plate with the mTeSR-1 medium. The cells generated from an individual colony were defined as the individual iPS cell line and used for further experiments.Retroviruses were produced as described previously . BrieflyThe iPSCs were fixed with 4% paraformaldehyde for 15 min at room temperature. Then, cells were stained with Naphthol phosphate  in Fast Red  that was prepared in Tris-HCl (pH 9.2) for 30 min in the dark at room temperature. Images were taken randomly by a microscope .7 cells resuspended in 100 μl DMEM/F12, and 200 μl of Matrigel. Cells were injected into the dorsal flank of a SCID mouse. Ten weeks after injection, tumors were collected and fixed with 4% paraformaldehyde. Subsequently, paraffin-embedded tumors were sectioned and stained with hematoxylin and eosin.To form teratomas, the iPSCs were harvested by 0.5 mM EDTA, 10Karyotype analyses were performed in Cytogenetic Laboratory, Huazhong University of Science and Technology. In brief, the cells were treated with 10 mg/ml colchicine for 60 min at 37 °C, harvested with 0.5 mM EDTA, exposed to 0.075 M KCl hypotonic solution for 30 min, and fixed five times with methanol/acetic acid (3:1). The cells were then analyzed by Giemsa-banding.4 cells/cm2 in non-adhesive culture dishes containing the mTeSR-1 medium supplemented with ROCK inhibitor Y27632 , which was added to promote the formation of EBs on the first day of the suspension culture. On day 2, the medium was replaced with 20% knockout serum replacer medium . After 1 week of suspension culture, the EBs were transferred to a standard culture dish and were cultured in MSC complete culture medium. When 90% confluence was reached, the differentiating cells were passaged at the ratio of 1:3, and the process was continued until the cells exhibited MSC-like morphology and analyzed for expression of MSC surface markers by flow cytometry weekly. After approximately six passages, the cells exhibited a typical fibroblast-like growth pattern and expressed MSC surface markers, and at this stage, the cells were referred to as the iPSC-MSCs (passage 1).The differentiation of the iPSCs into MSCs was initiated with the formation of embryoid bodies (EBs), as reported previously  with cerOsteogenic differentiation was induced with DMEM-L medium  supplemented with 10% FBS, 10 mmol/l β-glycerophosphate , 0.1 μM dexamethasone , and 50 μg/ml ascorbic acid . The medium was changed every 3 days. After 2 weeks, osteogenic differentiation was analyzed by Alizarin Red staining. Briefly, cells were fixed with 4% paraformaldehyde for 15 min, then stained with 40 mM Alizarin Red solution (pH 4.2)  for 10 min at room temperature, and washed with PBS until nonspecific staining. Images were taken randomly using a microscope. The quantitative analysis was performed using 10% cetylpyridinium chloride  extraction. Absorbance was measured at 560 nm. Experiments were repeated six times.For adipogenic differentiation, cells were cultured in DMEM-H medium  supplemented with 10% FBS, 0.5 mM isobutylmethylxanthine , 1 μM dexamethasone, and 10 μg/ml insulin . After 2Total RNA of cells was extracted using Trizol reagent (Invitrogen) through standard protocols according to the instructions of the manufacturer. The concentration and quality of total RNA were checked by NanoDrop 2000 (Thermo Fisher Scientific) and gel electrophoresis. Total RNA was reversely transcribed into cDNA. Real-time PCR was performed in the CFX Connect™ Real-Time PCR Detection System (Bio-Rad) using SYBR Premix Ex Taq (TOYOBO). The relative expression of mRNA was quantified using the ΔΔCt method . The pri4 cells were seeded in a 24-well-plate supplemented with 10 μM 5-Bromo-2-deoxyuridine  and cultured for 48 h. After fixation with 4% paraformaldehyde, BrdU incorporation was detected by fluorescent staining using an anti-BrdU antibody, and nuclei were stained with DAPI. The total number of cells was determined by counting DAPI-positive cells. Images were taken randomly by a fluorescence microscope . The percentage of proliferating cells was determined by dividing the number of BrdU/DAPI double-positive cells by the total number of cells. The proliferation assays were performed by more than 4 independent experiments, and each experiment randomly selected 4 images for statistics.Cell viability was detected at different times by cell counting kit-8 (Dojindo) according to the manufacturer’s instructions. Cells were seeded in the 96-well plate at a density of 8000 cells/well. The absorbance was measured at 450 nm, and the corresponding cell number was calculated according to the standard curve. Six to eight independent experiments were performed. BrdU labeling was performed to assess cell proliferation. 10In order to study the senescence of the BMSCs (Passage 4) and iPSC-MSCs (Passage 4), the Senescence Cells Histochemical Staining Kit  was used in accordance with the manufacturer’s instructions. Random images were photographed during observation under a microscope . The number of cells with intracellular blue deposits divided by the total number of cells was the ratio of senescent cells. Five independent experiments were performed to ensure reproducibility.β value (ranging from 0 to 1). The CpG sites with β value < 0.2 and > 0.8 were regarded as hypomethylated and hypermethylated sites, respectively. The number of beads per feature varying between chips and the β values was calculated as the average of at least three experimental replica values.Genomic DNA was isolated with the DNA Blood Midi Kit . DNA quality was assessed using a spectrophotometer and gel electrophoresis. The DNA samples (500 ng) were bisulfite converted using the EZ DNA Methylation Kit  in accordance with the manufacturer’s instructions. The DNA methylation profiles were analyzed using Infinium HumanMethylation850 BeadChip (Illumina) in accordance with the manufacturer’s instructions. The initial analysis was performed in Genomestudio 2010.3 (Modul M Version 1.8.5) at the Shanghai Biotechnology Corporation . The obtained data were normalized with the internal controls according to Illumina’s standard procedures. The level of methylation at each locus was calculated using the GenomeStudio Methylation module as the A total of 40 adult male Sprague Dawley rats were used for the study. The experiments were performed in the Animal Experiment Center of Tongji Medical College, Huazhong University of Science and Technology. All procedures were conducted in strict accordance with the guidelines for the Care and Use of Laboratory Animals.n = 10 per group): (a) normal control group, (b) methylprednisolone (MPS) group, (c) MPS + BMSC-treated group, and (d) MPS + iPSC-MSCs-treated group. ONFH was induced in the relevant groups of rats using MPS, as described previously [A total of 40 male Sprague Dawley rats (8–10 weeks old and weighing 250–300 g) were purchased from the Animal Experiment Center of Tongji Medical College, Huazhong University of Science and Technology. The rats were assigned randomly to the following groups (eviously  with sli7 cells/ml) or iPSC-MSCs (107 cells/ml) was injected. After injection, the needle was left in the tissue for 1 min before slowly withdrawing, and the marrow cavity was sealed with sterilized bone wax. Animals returned to their homes for standard husbandry. The rats of the BMSC-treated group, and the iPSC-MSC-treated group were injected with BMSCs and iPSC-MSCs, respectively. The MPS group rats were injected with 0.2 ml PBS. The duration of the intervention was 8 weeks. The femoral head samples were obtained after the rats were sacrificed ethically.Cell labeling and transplantation were performed as previously described , 40, 42.Micro-CT analysis was performed at week 4 and week 8 after cell implantation, using SkyScan 1176 scanner . The femoral heads were fixed with 4% paraformaldehyde and subsequently analyzed under SkyScan1176 scanner. The scanner was set at a resolution of 9 μm per pixel. The images were reconstructed and analyzed using the NRecon software . The region of interest (ROI) for the analysis and comparison of the trabecular parameters was determined based on a previous study . The traTo perform the immunofluorescence analysis, the cells were fixed with 4% paraformaldehyde for 15 min, followed by washing with PBS and subsequent treatment with 5% bovine serum albumin  in PBS containing 0.1% Triton X-100 for 1 h. The primary antibodies used in the experiment were rabbit anti-human OCT4 , rabbit anti-human SOX2 , rabbit anti-human Nanog , mouse anti-human TRA1–81 , TRA1–60 , SSEA4 , mouse anti-CD31, mouse anti-VEGF , and mouse anti-BrdU . The secondary antibodies used were as follows: TRITC-conjugated goat anti-Mouse-IgG , FITC-conjugated goat anti-Mouse-IgG , TRITC-conjugated goat anti-rabbit IgG , and HRP-conjugated goat anti-Mouse-IgG . Cell nuclei were stained with DAPI . Images were acquired during observation under a fluorescence microscope .The harvested femoral head tissues were fixed with 4% paraformaldehyde, decalcified using 10% EDTA solution (pH 7.4), dehydrated, and finally embedded in paraffin. The paraffin-embedded sections were then stained with hematoxylin-eosin  as previously reported . For Mast tests were used to compare the means of two different groups. One way-ANOVA was performed for multiple comparisons, with p < 0.05 as the threshold of statistical significance. All experiments were repeated three times or more.Statistical analysis charting was performed in GraphPad Prism 7.0 software . All results were presented as mean ± SEM unless otherwise stated. Independent-sample MSCs were isolated from the bone marrow samples of the ONFH patients (ONFH-BMSCs), and the culture was expanded for three passages. As depicted in Figure SAfter the establishment of the iPS cell line, the iPSCs were differentiated into MSCs through induction of embryoid body (EB) formation Fig. a. After p < 0.0001), which was consistent with a previous report [p < 0.01) and similar to that in the control group . Moreover, after the reprogramming, the number of lipid vacuoles in the iPSC-MSC group was significantly lower than that in the ONFH-BMSC group  and equivalent to that in the control group , while a higher proliferation rate was observed for iPSC-MSCs compared to ONFH-BMSCs . After reprogramming, the percentage of senescent cells in the iPSC-MSC group was lower compared to both ONFH-BMSCs group  and control group , while the iPSC-MSCs could inhibit PHA-stimulated lymphocyte proliferation, which indicated that the iPSC-MSCs possessed certain immunomodulatory functions , and no tumor formation was observed in these SCID mice even after 3 months of inoculation with iPSC-MSCs. Collectively, these findings indicated that the iPSC-MSCs exhibited faster proliferative capability, immunoregulatory function, and low immunogenicity, without tumor formation.Currently, MSCs are the most preferred cells for cell replacement therapy. One of the main advantages of MSCs is their low immunogenicity and a certain capacity for immunoregulation. Therefore, MSC transplantation can reduce immunological rejection to a certain extent. In this context, immunogenicity and the immunoregulatory function of the generated iPSC-MSCs were assessed based on the detection of immune-related surface molecules (CD80 and CD86) and co-culture with rat lymphocytes. Costimulatory molecules CD80/CD86 play an important role in the activation, proliferation, and immune response of T lymphocytes. FACS results revealed almost nil expression of CD80 and CD86 (< 1%) in the iPSC-MSCs, similar to BMSCs Figure Sa, b. It  Figure Sc. The rep value < 0.05) : normal BMSCs implant group (MPS + BMSCs), iPSC-MSCs implant group (MPS + iPSC-MSCs), MPS group (MPS + PBS), and normal control group. The intramedullary injection was performed 8 weeks after the induction of the ONFH model. Observation upon 4 weeks after the intramedullary injection revealed an uneven distribution of BrdU-positive cells in the bone marrow cavity, cartilage, and the subchondral area of the femoral head, as well as in trabecular bone, as evidenced by immunohistochemistry. Eight weeks after the injection, a further increased number of BrdU-positive cells could be observed in the bone marrow cavity and trabecular bone , as described previously , 43. Thep < 0.01) 8 weeks after the implanting. In addition, Tb.Sp was remarkably increased in the MPS group compared to the control group, and this increase could be profoundly inhibited by implanting BMSCs or iPSC-MSCs  was selected in the femoral head for observation, and statistical analysis was performed as described previously . In the SCs Fig. b.Fig. 5The histological evidence based on HE staining revealed sparse trabecular bone, which even disappeared in certain areas, empty lacunae in the cavities of trabecular bone, and decreased osteocytes in the MPS group Fig. c. Contrap < 0.0001) microvessels of the subchondral bone, which was higher than that in the BMSC implant group (p < 0.05) and similar to that in the control group (p = 0.54)  Fig. b. These MSCs have demonstrated considerable prospects in the preclinical studies concerning ONFH treatment , 49, 50.The emergence of the cellular reprogramming technology has enabled generating iPSCs from autologous histiocytic cells, which has resolved the issues of limited cell sources and immune rejection in cellular therapy, introducing novel strategies for regenerative medicine, disease models, and drug research and development. The selection of donor cells forms the basis of cellular reprogramming. Although it is possible to reprogram human skin fibroblasts into iPSCs , BMSCs wIt was demonstrated that BMSCs are a suitable source for reprogramming, allowing the successful establishment of patient-specific iPSCs. The iPSCs generated in our study exhibited the characteristics of completely reprogrammed cells, including ESC-like colony morphology, alkaline phosphatase expression, and the expression of pluripotent markers such as Oct4, Sox2, Nanog, SSEA4, TRA-1–60, and TRA-1–81  for use as the standard definition of human MSCs . The resTo facilitate tracking of the transplanted MSCs, the MSCs were labeled in vitro before transplantation. GFP labeling is an easy-to-observe widely-used labeling method . HoweverIn summary, the present study is a pioneer in demonstrating the equivalence of iPSC-MSCs and BMSCs concerning the treatment of steroid-induced ONFH in SD rats. In addition, it was demonstrated that the abnormal biological function and DNA methylation patterns of BMSCs in ONFH patients could be reversed by reprogramming these cells into iPSC-MSCs. The generated iPSC-MSCs exhibited morphology, immunophenotype, and in vitro differentiation potential analogous to normal BMSCs while exhibiting higher proliferation rates. Furthermore, iPSC-MSC implanting could effectively promote bone repair and angiogenesis in the necrosis area of the femoral head. Generating reprogrammed iPSC-MSCs represents an effective approach to provide a stable and reliable source of therapeutic cells, which could provide a novel direction of standardized MSCs to the treatment of ONFH in the future.Additional file 1: Figure S1. Flow cytometric analysis for the cell surface markers of BMSCs at passage 3. Figure S2. Karyotype analysis. The karyotype of iPSC was normal  after 37 passages. Figure S3. The immunogenicity and immunoregulatory function of MSCs. Flow cytometric analysis for immune-related surface molecules (CD80/CD86) of BMSCs (a) and iPSC-MSC (b). c Activity of iPSC-MSCs and BMSCs on the proliferation of stimulated T cells. Figure S4. Immunohistochemical staining of BrdU at the 4 weeks and 8 weeks after cell implantation. Scale bar indicated 50 μm. Figure S5. Gene expression of osteogenic gene Collagen type I and ALP after two weeks of osteogenic induction. For each gene, expression levels were normalized to the control group, n = 4 per group. All data are presented as mean ± SEM. One-way ANOVA with Tukey’s adjustment . Table S1. Clinical characteristics of recruited patients. Table S2. Sequence of RT-qPCR primers.


A) receptors hyperactivities that persisted even the anesthetic compounds have been eliminated. Here, we investigated the antidepressant, venlafaxine (VLX), in a mouse model of POCD and studied whether VLX attenuated the cognitive dysfunction of mice exposed to general anesthetic, isoflurane (ISO). We found that ISO significantly induced an increased surface expression of the GABAA receptor subunit, α5, in the hippocampus of the mice. However, VLX treatment reduced the increase in α5 subunit expression. Meanwhile, we found the expression levels of interleukin (IL)-1β, tumor necrosis factor alpha (TNF-α), and IL-6 in the brains of mice exposed to ISO were significantly increased. However, VLX could prevent the increase in these cytokines. We also investigated the memory deficit of these mice by using a Y maze behavioral test. Mice with ISO exposure showed decreased alternation performance that could be prevented by the VLX treatment. Collectively, our results here are in line with the previous findings that α5 subunit plays an important role of the formation of POCD, but VLX may be a promising candidate compound for the treatment of POCD.Post-operative cognitive dysfunction (POCD) is a common complication during the post-operative period. It affects the recovery time of the patient after surgery and the stay time in hospital, which causes a great deal of burden to patients and families emotionally and financially. However, there is no specific and effective treatment available for this disorder. Recent study indicated exposure to general anesthetics contributed to POCD by triggering gamma-amino butyric acid type A (GABA Post-operative cognitive dysfunction (POCD) is featured with cognitive decline, such as memory and executive functions after surgeries, including thyroid cancers, especially in elderly patients. It could last days, weeks, and months even longer than several years . StatistARs) are critical inhibitory receptors, and most general anesthetics are positive allosteric modulators of GABAARs by increasing the inhibition in the brain, which causes amnesia and loss of consciousness , gamma-amino butyric acid type A receptors  is one of the widely used antidepressants due to its relatively safe profile and efficacy on depressive and anxiety disorders . Study aIn the present study, we investigated the possible effect of VLX on the prevention of POCD in mice exposed to ISO. We found that ISO could cause decreased spontaneous alternation of mice in a Y maze test that is a behavioral assay of working memory , but VLXAll procedures involving animals were approved by the Experimental Animal Ethics Committee of China–Japan Union Hospital of Jilin University, Jilin Province, China. Male C57BL/6 mice at the age of 12 weeks old were used in the present experiments. Mice were kept in the standard laminal-flow cabinets with pathogen-free conditions. Mice were randomly grouped into multiple groups: control group, venlafaxine alone group (VLX), ISO alone group (ISO), and ISO plus venlafaxine group (ISO + VLX). VLX was purchased from Sigma-Aldrich and dissolved in normal saline. For the ISO treatment, the mice were anesthetized with inhalation of 4% ISO for the induction phase and then 1.3% for 3 h in a chamber on a heating pad to keep body temperature with 100% oxygen . The breMice were sacrificed on the day when the behavioral tests were completed. After perfusion with 0.9% normal saline, the hippocampal tissues from the mice brains were collected for the determination of protein concentration with Western blot, ELISA, and biotinylation.Working memory was assessed in a Y maze apparatus consisting of three arms, which was a 1-day test. The Y maze test was carried out for 3 days and 1 week later after the anesthesia. Each arm of the Y maze was 35 cm long, 25 cm high, and 10 cm wide and positioned at an equal angle as previously reported . The thrTo measure the protein expression level of p-STAT3 and STAT3, tissues from the hippocampus of mice were lysed with radioimmunoprecipitation assay (RIPA) buffer and then centrifuged at 12,000 g at cold condition. The supernatant containing equal amount of total protein was separated in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel for 2 h. After transferring to polyvinylidene fluoride (PVDF) membranes and blocking with 5% bovine serum albumin (BSA) in Tris-buffered saline with Tween 20 (TBST) buffer, the membranes were incubated with primary antibodies of p-STAT3 , STAT3 , and β-actin  in cold room for overnight and then in secondary antibodies for 2 more hours at room temperature after washing with TBST. Chemiluminescence with enhanced chemiluminescence (ECL) solution was used to visualize the signal from the membranes. The expression level of each protein in statistical analysis was presented as the relative ratio compared to the internal control protein, β-actin.Cytokines were measured with the commercial ELISA kits (Thermo Scientific) according to manufacturer's protocols, respectively. Briefly, the total protein was isolated from hippocampal tissues as above mentioned in the Western blot, and the supernatant was used for ELISA assay after concentration was determined. All samples were assayed with duplication. Standard curve for each cytokine was established with their standard samples provided in the kits. The values of these cytokines were normalized as a relative ratio compared to the total protein.2/5% CO2. The slices were then incubated twice in ice-cold 0.75 mg/ml NHS-SS-biotin (Thermo Scientific) for 45 min. Then, slices were washed several times to stop the excess biotin reaction followed by adding lysis buffer with protease inhibitors. Hi-Capacity NeutrAvidin beads (Thermo Scientific) was used to incubate supernatant for 16–18 h at 4°C to pull down and elute the proteins with elution buffer. The protein concentration was determined, and equal amount protein was subjected to SDS-PAGE analysis as Western blot assay. GABAA receptor α5 subunit antibody , β-actin antibody , and anti-Na+/K+ ATPase antibody  were used for Western blot analysis. The expression of α5 was normalized to its respective loading controls in the surface (NKA) and total (β-actin). The purity of extracted surface protein was determined by probing the presence of β-actin with antibody.Cell-surface biotinylation was carried out as previously reported . Brieflypost-hoc test for multiple comparisons. A p < 0.05 was deemed to show statistical significance.All statistical analysis was completed with Prism software. Data were expressed as the mean ± SEM. The significance of differences was determined by two-way ANOVA, followed by Tukey's F1, 44 = 14.12, p < 0.001] and VLX  exerted a significant change in the alternation performance. There was an interaction between ISO and VLX  in day 3. A post-hoc analysis indicated that the alternation behavior in the ISO-treated mice was less than that of the control mice (p < 0.001), and VLX significantly prevented the reduction in the alternation performance (p < 0.01) , and VLX significantly prevented the reduction in the alternation performance (p < 0.01)  . Interes < 0.01) . Moreove < 0.01) . On the  < 0.01) . ISO and < 0.01) . These aF1, 20 = 44.60, p < 0.0001] and VLX  exerted a significant change in the protein level of p-STAT3. There was an interaction between ISO and VLX  on the change of p-STAT3 protein. A post-hoc analysis indicated that p-STAT3 protein in the ISO-treated mice was higher than that of the control mice (p < 0.0001), and VLX significantly prevented the increase in the p-STAT3 protein (p < 0.01)  . These aF1, 24 = 24.80, p < 0.0001] and VLX  exerted a significant change in the IL-6 protein level. There was an interaction between ISO and VLX  on the changes of IL-6 protein level. A post-hoc analysis indicated that the IL-6 protein level in the ISO-treated mice was higher than that of control mice (p < 0.0001), and VLX significantly prevented the increase in the IL-6 protein level (p < 0.01) , and VLX significantly prevented the increase in the IL-1β protein level (p < 0.01)  . For IL- < 0.01) . For TNF < 0.01) . These rA receptor α5 was shown to be responsible for the changes in tonic current that was considered as the molecular mechanism of memory impairment in POCD , and VLX significantly prevented the increase in the surface expression level of α5 (p < 0.01)  . There w < 0.01) , which sPOCD happens frequently following major surgery. The incidence of this disorder is around one-third in major surgeries and much higher in elderly patients. However, there is no specific treatment option for it yet.A receptor α5 in the anesthetics-induced POCD. The mouse model used here has been verified by several recent studies from other groups by using single dose of etomidate, which caused memory and cognitive impairment, POCD  of venlafaxine could lower the threshold of convulsion in animals and suggested the use of 25 and 50 mg/kg for animal study . AnotherIt has been well-established that tissue trauma released damage-associated cytokines, including TNF-α, could break the blood–brain barrier (BBB) and result in neuroinflammation and concomitant cognitive decline . MoreoveA receptor α5 subunit plays a key role in POCD  antidepressant, VLX has potent effect on serotonin receptor and increase the content of serotonin. Serotonin has been found to be a modulator of GABA neurotransmission . Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Coronavirus disease 2019 (COVID-19) is an infectious disease caused by a novel coronavirus that has spread rapidly, resulting in a worldwide pandemic. Even though end-stage renal disease (ESRD) patients are particularly susceptible to COVID-19 infection and can develop severe to critical disease, there are limited studies and case reports about COVID-19 in ESRD patients. We report a case of a 63-year-old gentleman with ESRD on regular hemodialysis. We describe the clinical presentation of this patient, the diagnostic process, the laboratory and imaging investigations, as well as the course of treatment. He positively responded to a 14-day course of Lopinavir-Ritonavir, Ribavirin, Azithromycin, and Hydroxychloroquine. In December 2019, a novel coronavirus was recognized as the cause of a group of pneumonia cases in Wuhan, a city in the Hubei Province of China. It quickly spread, resulting in an epidemic throughout China, followed by a worldwide pandemic with almost 2 million confirmed cases. In February 2020, the World Health Organization (WHO) named the disease COVID-19, which stands for coronavirus disease 2019. The virus that causes COVID-19 was named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [Although severe COVID-19 disease can occur in otherwise healthy individuals of any age, it predominantly affects adults with advanced age or underlying medical co-morbidities , 3. End-We report a case of an ESRD patient on regular hemodialysis with severe COVID-19 pneumonia. This report describes the clinical presentation of this disease in a hemodialysis patient, the diagnostic process, the laboratory and imaging investigations, as well as the course of treatment.A 63-year-old hemodialysis patient presented with a history of productive cough with whitish sputum and worsening shortness of breath for four days duration. His shortness of breath was not associated with orthopnea or paroxysmal nocturnal dyspnea. His wife was confirmed to have COVID-19 disease three days before his symptoms started. He denied any history of fever, sore throat, nasal congestion, or headache. He was later admitted in a special isolation hospital designated for COVID-19/ suspected COVID-19 patients in Bahrain. He has a medical history of hypertension, diabetes mellitus type 2, ischemic heart disease, and ESRD. He has been on hemodialysis three-times-weekly through a tunneled vascular catheter. His regular medications include Sevelamer, Calcitriol, Rosuvastatin, Amlodipine, and Perindopril. His blood sugar is controlled without medications. Based on the Ministry of Health in Bahrain guidelines, the hemodialysis facility where the patient was receiving his scheduled dialysis took specific precautions to reduce transmission and protect the patients. The facility was disinfected and the patient’s close contacts  were traced, tested for the virus, and asked to self-isolate for 14 days . All his2 / FiO2 ratio of 322). On chest examination, he had symmetrical breath sounds bilaterally, with coarse inspiratory crackles that were more prominent on the right side. His cardiovascular examination showed normal heart sounds with no murmurs or added sounds and his jugular venous pressure was not raised. He did not have peripheral edema. His abdominal and neurological examinations were unremarkable.On admission, the patient was vitally stable, afebrile and was maintaining an oxygen saturation of 85% on room air. Although he was able to lie flat, he had labored breathing and his respiratory rate was approximately 25 breaths per minute. He was kept on a face mask with an oxygen flow of 6-7 L / min , and 2.2% eosinophils. His hemoglobin was 8.3 g/dl and his platelet count was 113 X 109 per L. His urea was 18.4 mmol/L and his creatinine was 716 µmol /L. His sodium was 138 mmol/L, potassium 4.7 mmol/L , chloride 99 mmol/L, bicarbonate 26 mmol/L, calcium 2.01 mmol/L, phosphorus 2 mmol/L and magnesium 0.8 mmol/L. C-reactive protein (CRP) and procalcitonin (PCT) were 20.9 mg/L (Reference range 0-3 mg/L) and 0.58 µg /L (reference range 0-5 µg /L) respectively. His ESR was 45 mm/hour, Serum ferritin 2070 µg /L, and D-dimer 22.12 mg/L. His liver function tests and cardiac enzymes were within the normal range.Laboratory tests on admission showed a white blood cell count of 3.23 X 102 / FiO2 ratio 250). An urgent portable chest x-ray revealed bilateral patchy lower lobe predominant airspace opacification more diffusely involving the right lung, and more peripherally in the left lung  was done every alternate day to monitor the QT interval on antibiotic therapy which did not exceed 450 msec. His ECG showed baseline findings including a first-degree Atrioventricular block with poor R-wave progression, and no acute ischemic changes were noted. His repeated echocardiogram on this admission did not reveal any new findings. He had a left ventricular ejection fraction of 35 -40% with persistent regional wall motion abnormalities noted in septal and apical regions. His central and peripheral blood cultures were sterile on two separate occasions. He also had a sterile endotracheal aspirate culture.The patient responded gradually to medical therapy and his oxygenation continued to improve until he was successfully weaned from oxygen. He continued to receive his hemodialysis sessions as scheduled bedside. His fluid balance was monitored closely, and adequate ultrafiltration was ensured. His blood count, electrolytes, and ECG findings remained consistent during the hospital stay.After 14 days in the high dependency unit, his condition improved clinically, and he was shifted out to the ward under isolation settings. A repeated SARS-CoV-2 virus nucleic acid swab was negative. Although the patient's radiographic findings lagged behind the improvement in clinical condition, his repeated chest x-ray showed partial interval resolution of the opacities which was more evident in the left lung and upper zone of the right lung . He was labeled as suspected case based on having close contact with a confirmed COVID-19 case and presenting with respiratory symptoms, imaging features suggestive of COVID-19 pneumonia as well as lymphopenia on admission. The diagnosis was confirmed by identifying SARS-CoV-2 virus nucleic acid by Real Time Reverse-Transcription Polymerase Chain Reaction (RT-PCR). The case was further classified as a severe disease due to having an oxygen saturation below 93% at rest on admission and having more than 50% lung involvement on imaging within 24 hours [Our patient exhibited laboratory findings common among COVID-19 patients , 9. He hCommon abnormal radiographic findings in the chest x-rays of COVID-19 pneumonia patients were consolidation and ground-glass opacities, with bilateral, peripheral, and lower lung zone distributions. Our patient had typical chest x-ray findings (Figure The patient management plan was based on evidence-based medicine as well as the protocols published by the Ministry of Health in Bahrain for the treatment of severe COVID-19 pneumonia. The COVID-19 treatment approach remains uncertain with no therapies clearly proven effective. In addition to supportive care, the patient was started on a COVID-19 regimen composed of Lopinavir-Ritonavir, Ribavirin, Azithromycin, and Hydroxychloroquine . The dosThe evidence on the possible efficacy of the proposed regimen for severe COVID-19 pneumonia is largely from case reports and observational studies. Lopinavir-ritonavir and Ribavirin have been used successfully as monotherapies in the treatment of mild COVID-19 pneumonia in hemodialysis patients , 10. In Empiric antibiotic therapy for possible co-infection was also initiated. The choice of empiric antibiotics was based on clinical judgment, the bacterial pathogens commonly isolated at the patient’s hemodialysis facility, and his own bacterial culture history. Oseltamivir was added to empiric therapy because influenza pneumonia was included in the initial differential diagnosis based on clinical presentation and chest x-ray findings.After discharging the patient, he will be in home quarantine for another 14 days. He will be tested again for the virus at day 7 and day 14 post-discharge. He will also be monitored closely for any respiratory and gastrointestinal symptoms. Meanwhile, precautions will be taken to isolate the patient in the dialysis facility and minimize his contact with the healthcare staff. Safety measures will also be taken in transporting the patient to and from the dialysis facility.ESRD patients with COVID-19 can present atypically and the index of suspicion should be high in the setting of a pandemic to avoid exposure to healthcare staff and other hemodialysis patients. COVID-19 therapy in ESRD is especially challenging due to the requirement of renal dose adjustment of various medications and the propensity of ESRD patients to develop QT prolongation due to concomitant hypocalcemia and other electrolyte imbalances. Further clinical trials and observational studies are required to clearly understand the whole spectrum of clinical presentations and the optimal diagnostic and treatment methods for COVID-19 disease in hemodialysis patients.


Necrotizing enterocolitis (NEC) is the leading cause of death due to gastrointestinal disease in preterm infants. The role of miRNAs in NEC is still unknown. The objective of this study was to identify differentially expressed (DE) miRNAs in rats with NEC and analyze their possible roles. In this study, a NEC rat model was established using Sprague-Dawley rat pups. Small RNA sequencing was used to analyze the miRNA expression profiles in the NEC and control rats. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were carried out to identify target mRNAs for the DE miRNAs and to explore their potential roles. The DE miRNAs were verified by real-time quantitative PCR (RT-qPCR). The status of intestinal injury and the elevated levels of inflammatory cytokines in the NEC group confirmed that the NEC model was successfully established. The 16 miRNAs were found to be differentially expressed between the NEC group and the control group of rats. Bioinformatics analysis indicated that the parental genes of the DE miRNAs were predominantly implicated in the phosphorylation, cell migration, and protein phosphorylation processes. Moreover, the DE miRNAs were mainly found to be involved in the pathways of axon guidance, endocytosis, and focal adhesion, as well as in the Wnt signaling pathway, which is related to colitis. The expression patterns of the candidate miRNAs (rno-miR-27a-5p and rno-miR-187-3p), as assessed by RT-qPCR, were in accordance with the expression patterns obtained by miRNA-sequencing. The miRNA/mRNA/pathway network revealed that rno-miR-27a-5p and rno-miR-187-3p might be involved in NEC via the Wnt signaling pathway. We found an altered miRNA expression pattern in rats with NEC. We hypothesize that rno-miR-27a-5p and rno-miR-187-3p might mediate the NEC pathophysiological processes via the Wnt signaling pathway. Necrotizing enterocolitis (NEC) is considered one of the leading causes of neonatal morbidity and mortality . NEC is miRNAs are small noncoding RNAs of 18-24 nucleotide length. Abnormal expression of miRNAs is associated with human cancer and diseases . A varieThe primary objective of this study was to analyze the miRNA expression profiles in rats with NEC. To achieve this, we established an NEC rat model and then sequenced the intestine of these rat pups using NGS technology to identify differentially expressed (DE) miRNAs between the NEC animals and the control animals. We then constructed a DE miRNA and target mRNA regulatory network to recognize the potential functions of these DE miRNAs in NEC.All animal experiments were approved by the Institutional Animal Ethics Committee of Wuxi Maternity and Child Health Care Hospital and carried out in compliance with national and international guidelines for the Care and Use of Laboratory Animals. Major efforts were made to minimize the suffering of the Sprague-Dawley rat pups used in this study.μg/g lipopolysaccharide (LPS) after 8 h of birth, once per day. After LPS, animals were subjected to hypoxia and hypothermia twice a day for two consecutive days. Briefly, animals were exposed to hypoxia in a Plexiglass chamber infused with 100% nitrogen for 60 s, immediately followed by exposure to 4°C for 10 min. The severity of NEC in the rats was assessed based on the NEC scoring system [The NEC model was developed as described by Wang et al. . On the g system . Grade 0μm thick sections. After H&E staining, a histopathological examination was performed, and the histopathological condition of the intestine tissues of rats in each group was observed and documented with photographs.The 1.0 cm sections of ileocecum tissues were fixed in 4% paraformaldehyde and then embedded in paraffin. Hematoxylin and eosin (H&E) staining was performed on 4 α and IL-1β in the control and NEC groups were measured using real-time quantitative PCR (RT-qPCR). TRIzol reagent  was utilized to extract the total RNA, according to the manufacturer's protocol. The total RNA was reverse-transcribed with a commercial reverse transcription kit , followed by RT-qPCR (2x Master Mix kit from Roche), according to the manufacturer's instructions. FastStart Universal SYBR Green Master mix was used to amplify cDNA on the QuantStudio 6 Flex Real-Time PCR System .The mRNA levels of TNF-The small RNA library was constructed using the Multiplex Small RNA Library Prep Kit for Illumina method , according to the manufacturer's instructions. Following RNA quality control, index codes were added to attribute sequences to each sample. Next, serial processes including 3′ adaptor ligation reaction, RT primer hybridization, 5′ adaptor ligation reaction, and double-stranded cDNA synthesis were performed. The RNA samples were sequenced by Shanghai Yingbai Biotechnology Co. Ltd. using the HiSeq 2500 platform.http://www.mirbase.org/) to identify known miRNAs (20-25 bp). The raw counts were used as input for the EBSeq algorithm, and DE miRNAs were identified following the criterion  < 0.05); FDR was calculated to correct the p value.The raw sequences were filtered and optimized to remove short (<15 nt) and low-quality reads. The clean reads were then matched to the miRBase database (http://www.microrna.org/microrna/home.do) was employed to predict target mRNAs of the DE miRNAs (score ≥ 150 and energy < −20). Subsequently, the RNAhybrid algorithm (https://bibiserv.cebitec.uni-bielefeld.de/RNAhybrid/) was also applied to predict the target mRNAs of the DE miRNAs (energy < −25). The intersection of the two algorithms was taken as the final result.The miRanda algorithm  and subjected to reverse transcription by PrimeScript™ RT reagent kit . RT-qPCR was carried out using the ABI Q6 detection system . GAPDH was used as the endogenous control. The 2p values were calculated using GraphPad Prism 8. Student's t-test was used for comparisons among groups. p < 0.05 was considered statistically significant.Data from three independent experiments was expressed as mean ± SD. The α and IL-1β, were found to be higher in the NEC group than in the control group  and the 1 downregulated miRNA (rno-miR-187-3p) were verified by RT-qPCR. Thus, the expression patterns of these five DE miRNAs, as analyzed by RT-qPCR, were in accordance with the expression patterns obtained by miRNA sequencing. However, only the expression patterns of rno-miR-27a-5p and rno-miR-187-3p were found to be statistically significant .We used Cytoscape software to depict an integrated mRNA/miRNA interaction network that included the upregulated miRNA, rno-miR-27a-5p, and the downregulated miRNA, rno-miR-187-3p. Bioinformatics analysis showed that the target mRNAs of these two miRNAs were enriched in the Wnt signaling pathway. In this complex network, the target genes for rno-miR-27a-5p were Prkca, Plcb3, Vangl1, and Sfrp1, while the target genes for rno-miR-187-3p were Prkca, Ppar, and Prkcb .NEC is a high mortality disease that is responsible for 1–3 deaths per 1000 births per year among newborn infants in North America , 19. To γ signaling, leading to IL-12 expression in dendritic cells, thereby inducing Th1 differentiation in colitis [β signaling pathway has been shown to impair the healing of murine intestinal mucosal ulcers [In 2008, the first study to report miRNA expression patterns using tissue samples from inflammatory bowel disease patients was published . A study colitis . It is s colitis . Smad3-dl ulcers . In the β-catenin signaling pathway is essential for intestinal renewal, deregulation of which results in impairment of intestinal epithelial stem cell proliferation and differentiation [β-catenin signaling leads to defective intestinal regeneration in mice and humans with NEC. Exogenously administered Wnt7b has the potential to maintain intestinal epithelial homeostasis and avoid NEC intestinal injury [α and IFN-γ, can synergistically drive epithelial barrier dysfunction and apoptosis, particularly during colitis [α and IL-1β were found to be higher in the NEC group than in the control group. These results reveal that in rats with NEC, the occurrence of intestinal inflammation may be regulated by Wnt signaling.The pathogenesis of NEC is characterized by intestinal inflammation and injury. Previous reports state that the Wnt/ntiation . For insl injury . In the l injury . The pro colitis . Wnt5A i colitis , 31. In In conclusion, we identified 16 DE miRNAs between the NEC group and the control group. The target genes of the significant DE miRNAs were found to be enriched in the Wnt signaling pathway. We hypothesize that the identified miRNAs might be potentially involved in inflammatory response through the Wnt signaling pathway. In our future studies, we wish to further explore and verify this hypothesis.


Schizosaccharomyces pombe. As opposed to many other alkylating agents, ENU only induces an SN1-type reaction with a low s constant (s = 0.26), attacking preferentially OForward genetics in model organisms has boosted our knowledge of the genetic bases of development, aging, and human diseases. In this experimental pipeline, it is crucial to start by inducing a large number of random mutations in the genome of the model organism to search for phenotypes of interest. Many chemical mutagens are used to this end because most of them display particular reactivity properties and act differently over DNA. Here we report the use of N-ethyl-N-nitrosourea (ENU) as a mutagen in the fission yeast  The search of a particular phenotype of interest after natural or induced random mutation in biological models and the identification of the underlying genetic variant it is known as ’forward genetics’. This strategy represents a powerful tool for characterizing individual gene functions as well as complex genetic interactions when two or more mutations co-occur in the same cell. Human genetic diseases are usually provoked by hypomorphic mutations  that allow embryo development and birth but become deleterious at later stages. Therefore, by inducing mutations in genetic models, even in single-celled organisms such as yeasts, the genetic bases of human diseases can be closely reproduced for further investigation . Anothers). This is a measure of the sensitivity of the alkyl agent to the strength (n) of various nucleophilic reagents with a series of substrates in water solution (N1-type (low s) and SN2-type (high s). Most of the alkylating compounds used as mutagens range from s = 0.26 to s = 0.86. ENU causes an SN1-type reaction with a very low s constant (s = 0.26)   leading Standard fission yeast growth media and molecular biology approaches were used throughout (h reb1-GFP:hphMX6 gar2-mCherry:kanMX6), was grown in liquid yeast extract medium (YES) until log phase at 30°. The culture was harvested, washed twice with water and the cell density was adjusted to 10A prototroph reference strain (975 background), bearing G418 and hygromycin B resistance markers integrated into chromosomes I and II respectively . Respective open reading frames (ORF) were amplified by PCR. To identify base substitutions, the resulting products were sequenced using the Sanger method with forward and reverse primers, ensuring total coverage of both strands. As the wild type reference, we sequenced our parental strain markers to discard previously acquired polymorphisms relative to the database sequence.Genomic DNA was extracted from selected mutant strains , ENU only acts by a Ss constant (s = 0.26)  reduces viability in spite of such a short exposure time.The number of viable colonies in untreated ENU control was significantly and consistently much closer to the expected goal (500 colonies) than in the untreated EMS control . This suvs. EMS in fission yeast, we screened for auxotrophy-causing mutations as a gene loss-of-function readout for the same number of genomic targets. After carrying out the EMS and ENU mutagenesis described above  - and knockout of their orthologs in S. cerevisiae- become auxotrophic for histidine as well (ade9 and ade10), we checked out genetic linkage to csi1, which is just 8 Kb apart from ade9 (www.pombase.org). Neither mutation showed linkage to csi1 deletion marker, leaving ade10 as the only candidate. Consequently, ade10 locus was sequenced in both mutants and a different single base pair substitution was found in each strain . It is known that purine and L-histidine synthesis routes share a number of intermediates, which makes them interdependent . Furtherh strain , confirmade10-68 and ade10-424 attending to the protein residue affected. It is surprising that, among a limited number of mutations generated in this non-saturating mutagenesis, two of them were at the same locus. However, in a meta-analysis in mouse on the spectrum of ENU-induced mutations, Barbaric et al. found that genes with higher G-C content are more likely to be mutated by ENU and A-T pairs flanked by C or G bases are more prone to mutation (ade10 ORF contained the highest G-C percentage (46%) of the whole adenine pathway and significantly higher than the average G-C content (37.08%) of protein-coding DNA in fission yeast -5-aminoimidazole-4-carboxamide) as the substrate in the last steps of de novo purine biosynthetic pathway  . spAde10 pathway . There ihttps://fungi.ensembl.org/Schizosaccharomyces_pombe/Gene/Compara_Tree/pan_compara?g=SPCPB16A4.03c;r=III:947597-949685;t=SPCPB16A4.03c.1) revealed that valine is the most common residue in position 68  and it is present in most eukaryotes within a very well conserved motif   causes AICA-ribosiduria, a devastating disease characterized by severe neurological defects and congenital blindness. Abnormal accumulation of AICAR and defective purinosome formation is observed in the fibroblasts of these patients (Alignment of 170 orthologs recovered from the Ensembl database (ed motif . In posiquences) . In humapatients . Therefovs. ENU-induced loss-of-function mutation as well as to assess the procedure consistency, we repeated the protocol for a second screen   and 3. TE. coli, S. cerevisiae and C. elegans over 70% of changes correspond to G-C to A-T transitions while the remaining 30% are A-T to T-A transversions (Drosophila melanogaster germarium cells, proportions depend on pre- or post-meiotic stage (E. coli, S. cerevisiae or C. elegans. Again, this could be biased by the presence, specificity and efficiency of alkyltransferase and alkyltransferase-like protective proteins in S. pombe such as Atl1 against OUnlike other alkylating agents, indels imputed to ENU are extremely rare or absent in the literature in any model organism . The thr


Background: The Multidimensional Inventory for Religious/Spiritual Well-Being (MI-RSWB 48) was developed in order to address a religious/spiritual dimension as being an important part of psychological well-being. In the meantime, the instrument has been successfully applied in numerous studies. Subsequently, a short version, the MI-RSWB 12 was constructed, especially for the use in clinical assessment. Here it is intended to contribute to the further development of the MI-RSWB 12 by investigating its structural validity through structural equation modeling.Materials and Methods: A total sample of 1,097 German-speaking adults  from the normal population filled in the MI-RSWB 12 via an online-survey. In line with theoretical assumptions 5 different factor structure models for the MI-RSWB 12 were tested: (1) a single-factor model, (2) a model with four correlated RSWB dimensions, (3) a single higher-order model with four lower order factors, (4) a two higher-order model with four lower order factors, (5) a bifactor model, which includes four specific RSWB dimensions.Results: The single-factor model provided the poorest model fit, with no indices falling within the acceptable range. The four-factor, two higher-order factors and the bifactor models showed overall acceptable fit indices. With regard to the Akaike information criterion (AIC), the four-factor model demonstrated superiority compared to both the two higher-order factor model and the bifactor model, which in turn showed did not differ from each other.Conclusion: Four different MI-RSWB 12 sub-scales should be calculated in future studies, while a general factor and two higher order factors are statistically valid as well. Further applications of the MI-RSWB 12, especially in the clinical patient groups, are encouraged. In the past two decades in particular, there has been a remarkable increase in interest coming from psychology, medicine as well as the whole field of neurosciences in topics such as religiosity and spirituality as being related to various parameters of mental health, psychological well-being, and coping with illness  was developed based the original concept of the Spiritual Well-Being (SWB) scale  may be considered as a multidimensional alternative to the original SWB scale. Therefore, the MI-RSWB 48 was developed in order to meet the unanimous demand for a multidimensional assessment of religiosity and spirituality  differentiates between several religious  and spiritual dimensions , however does not consider a strict differentiation between an immanent and a transcendent area of perception ; Experiences of Sense and Meaning: “I have experienced true (authentic) feelings”; Hope Immanent: “I view the future with optimism”; Hope Transcendent: “I often think about the fact that I will have to leave behind my loved ones.” (coded reverse). The MI-RSWB 48 scale was used in a large number of studies in clinical as well as community samples .The sample was recruited through various social networks. Informed consent was acquired before each participant filled in the test form that included demographic questions as well as the standardized questionnaire described below. The data was acquired via the online-survey platform LimeSurvey. Data was analyzed from all participants that were aged at least 18 years, spoke German fluently and filled in all questionnaires. The study was carried out in accordance with the Declaration of Helsinki. Ethical approval was granted by the Ethics Committee of the Medical University of Graz, Austria.The Multidimensional Inventory for Religious/Spiritual Well-Being short version  > 0.90; (b) Tucker-Lewis Index (TLI) > 0.90; (c) the Normed Fit Index (NFI) > 0.90; (d) the square root error of approximation (RMSEA) < 0.08 and the upper bound of its 90% confidence interval < 1; and (e) χ2/df < 3. The χ2 significance test was neglected in this analysis as marker of good model fit, as a non-significant χ2 test is rarely obtained with large samples  was conducted with AMOS 26. SPSS 27.0 was used for data management, descriptive statistics and bivariate correlations. Goodness-of-fit was assessed with maximum likelihood (ML) estimation in AMOS. In accordance with Kline , the folAIC) was used, which rewards models that achieve a high goodness-of-fit and penalizes them if they become overly complex  A sM = 26.27; SD = 8.00). Most participants declared a general qualification for university entrance as their highest educational level . 396 (36.1%) participants declared a university degree as their highest educational level, 46 (4.2%) a high school degree and, 96 (8.8%) participants stated a completed apprenticeship as their highest educational level. 29 (2.5%) participants stated that they left school without graduation. Most participants stated to be religiously affiliated to Catholicism , 358 (32.6%) were without any affiliation, 55 (5%) were protestant, 33 (3%) were members of other Christian religious communities , while 16 (1.5%) identified themselves with other non-Christian religious communities . The nationality of most participants was either German , Austrian  or Swiss , while 50 (4.6%) stated other nationalities. In regard to their current relationship status, 71 (6.4%) were married, 518 (47.2%) in a relationship, and 508 (46.3%) were single.The investigated sample consisted of 1,097 German-speaking adults . The participants ranged in age from 18 to 69 years  was observed, while all other scales correlations remained insignificant (p > 0.001). No scale showed significant sex differences . Detailed descriptions of several scale characteristics can be retrieved from Regarding associations with age a small positive correlation with the MI-RSWB 12 scales GR , hence results where bootstrap corrected.r = 0.55–0.70; all p < 0.001). Furthermore, IWB was strongly related to HO  and FO , while GR  and CO  showed small correlations with IBW. This pattern was inversed for the TWB score: Large correlations were found with GR  and CO , while correlations with FO , and HO  were small.2/df = 46.61; RMSEA = 0.20 ; CFI = 0.54; NFI = 0.54; TLI = 0.44]. The single higher-order model showed generally acceptable fit , however exhibited a χ2/df > 3. In contrast, the four-factor , two higher-order factors  and the bifactor  models showed overall good fit indices, with all indices falling within the acceptable range. Every model exhibited a Bollen-Stine bootstrap p = 0.000. With regard to the AIC values, the four-factor model demonstrated superiority compared to both the two higher-order factor model (ΔAIC = 5.44) and the bifactor model (ΔAIC = 4.8), while no significant differences are observed between the bifactor and the two higher-order factor models (ΔAIC = 0.64).As shown in p < 0.01), except for the association between item 10 and the general factor (p > 0.05). The estimated strengths of the significant associations between the general factor and the individual items ranged from β = 0.22 to 0.74. Furthermore, the items assigned to the GR factor showed stronger factor loadings onto the general factor  than items assigned to the remaining dimensions of the MI-RSWB 12 (β = 0.22 to 0.54).The results of the CFA for the four-factor, two higher-order factors and the bifactor models, estimated with a bootstrap ML (250 samples), are detailed in r = 0.54; p < 0.01). Moreover, all assigned associations were significant . The higher-order factor TWB was substantially associated with both GR (β = 0.83) and CO (β = 0.79), while HO (β = 0.78), and FO (β = 0.38) were associated with IWB. Associations between the individual items and the dimensions of the MI-RSWB 12 ranged between β = 0.83 to 0.88 for GR, between β = 0.62 to 0.67 for CO, between β = 0.81 to 0.85 for HO, and between β = 0.47 to 0.82 for FO.Concerning the two higher-order factor model, both higher-order factors were substantially correlated  to r = 0.61 . All remaining correlations were significant .With regard to the four correlated factor model, the associations between the individual items and the MR-RSWB 12 dimensions remained unchanged. Correlations between the factors ranged from As detailed in It was intended in this study to further contribute to the flourishing research concerning the spiritual dimension within the area of psychological well-being, whereby we applied the Multidimensional Inventory for Religious/Spiritual Well-Being (MI-RSWB) in its original version with 48 items in various research settings. Based on these positive experiences a short version of the scale was constructed, especially for the application in clinical assessment. Hereby the original number of items were reduced from 48 items to 12 items (MI-RSWB 48 vs. MI-RSWB 12), as well as the originally six sub-dimensions were limited to four . Therefore, both sub-dimensions from the original MI-RSWB 48 scale: “Hope Transcendent” (HT) as well as “Experiences of Sense and Meaning” (SM) had to be deleted  were always shown to be less strongly linked to various parameters of mental health  compared to the parameters for Immanent Well-Being (HI and FO). In correspondence to this, according to Unterrainer , and UntIn terms of neural correlates of MI-RSWB, a voxel-based morphometry study regarding the belief in the miracles of Lourdes  dimension, which was also removed within the MI-RSWB 12. It is not for nothing that experiences of sense and meaning were extensively discussed within the context of nootherapeutic approaches (Viktor Frank and his Existential/Logotherapy can probably be mentioned here as the most prominent representative; Frankl, p-values for the χ2 statistic, adjusted standard errors and confidence intervals for parameter estimates. In correspondence, the significant Bollen-Stine bootstrap might be seen as problematic regarding the global fit of all investigated models (Bollen and Stine, A constraint of this study is the non-normality of the investigated data which might have affected the results of the CFA. Hence, Bollen-Stine bootstrap was employed to manage the effects of the violated multivariate normal assumption in terms of corrected So far, little has been done regarding the changeability of the RSWB dimensions over several measurement times. There is still a lot of work to be done here and a concise instrument seems to be preferable in many cases within a repeated measurement design. Based on the limited findings of previous research, it can be assumed that the RSWB dimensions can be changed in principle. However, these dimensions may need to be specifically addressed by means of a uniquely developed treatment protocol (e.g., Sollgruber et al., In conclusion, the MI-RSWB 12 short scale proves to be a very reliable instrument, with an excellent structural validity. Therefore, the MI-RSWB 12 is an adequate alternative for the long form of the scale (MI-RSWB 48; Unterrainer et al., human.unterrainer@univie.ac.at.The data analyzed in this study is subject to the following licenses/restrictions: Data set can be retrieved from the corresponding author. Requests to access these datasets should be directed to HU, The studies involving human participants were reviewed and approved by Medical University of Graz, Austria. The patients/participants provided their written informed consent to participate in this study.All statistical analysis were conducted by JF. JF wrote the first draft of the manuscript. HU read the manuscript and made some critical comments. JF and HU revised the whole manuscript together. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.